U.S. patent application number 10/168295 was filed with the patent office on 2005-03-24 for combination method for treating viral infections.
Invention is credited to Baroudy, Bahige M..
Application Number | 20050065319 10/168295 |
Document ID | / |
Family ID | 22973061 |
Filed Date | 2005-03-24 |
United States Patent
Application |
20050065319 |
Kind Code |
A1 |
Baroudy, Bahige M. |
March 24, 2005 |
Combination method for treating viral infections
Abstract
This invention provides novel combination therapies comprising a
CCR5 antagonist, or a pharmaceutically acceptable salt thereof, and
a DP-178 polypeptide, or a pharmaceutically acceptable derivative
thereof, for the treatment of HIV. The present combination permits
a more tolerable treatment schedule by reducing the frequency of
administration of a DP-178 polypeptide from twice daily
subcutaneously to once daily or even just a few times per week.
Inventors: |
Baroudy, Bahige M.;
(Westfield, NJ) |
Correspondence
Address: |
SCHERING-PLOUGH CORPORATION
PATENT DEPARTMENT (K-6-1, 1990)
2000 GALLOPING HILL ROAD
KENILWORTH
NJ
07033-0530
US
|
Family ID: |
22973061 |
Appl. No.: |
10/168295 |
Filed: |
June 19, 2002 |
PCT Filed: |
June 11, 2001 |
PCT NO: |
PCT/US01/18802 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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60256657 |
Dec 19, 2000 |
|
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|
Current U.S.
Class: |
530/324 |
Current CPC
Class: |
A61K 31/00 20130101;
A61K 2300/00 20130101; A61K 2300/00 20130101; A61K 31/00 20130101;
A61K 38/162 20130101; A61K 45/06 20130101; A61K 38/162 20130101;
A61P 31/18 20180101; A61P 43/00 20180101 |
Class at
Publication: |
530/324 ;
530/350; 514/002 |
International
Class: |
C07K 016/00; C07K
007/00; C07K 001/00; C07K 014/00; C07K 005/00; C07K 017/00; A01N
037/18; A61K 038/00 |
Claims
What is claimed is:
1. A method of treating an HIV infection in an individual in need
of such treatment, comprising administering in combination a
therapeutically effective amount of a CCR5 antagonist, or a
pharmaceutically acceptable salt thereof, and a therapeutically
effective amount of a DP-178 polypeptide, or a pharmaceutically
acceptable derivative thereof.
2. The method of claim 1, wherein the dosage of the CCR5
antagonist, or a pharmaceutically acceptable salt thereof, is 25 to
600 mg, and the dosage of the DP-178 polypeptide, or a
pharmaceutically acceptable derivative thereof, is 3 to 200 mg, or
a multiple thereof to reduce the viral load in the individual by 1
to 2 logs.
3. The method of claim 1, wherein the CCR5 antagonist, or a
pharmaceutically acceptable salt thereof, is administered 1 or 2
times per day of administration.
4. The method of claim 1, wherein the CCR5 antagonist, or a
pharmaceutically acceptable salt thereof, is administered from 1 to
3 times per week or every other day.
5. The method of claim 1, wherein the DP-178 polypeptide is
administered 1 to 3 times per week, or every other day.
6. The method of claim 1, wherein the DP-178 polypeptide is
administered 3 times per week or every other day.
7. The method of claim 1, wherein the CCR5 antagonist is
administered orally.
8. The method of claim 1, wherein the DP-178 polypeptide is
administered subcutaneously.
9. The method of claim 1, wherein the CCR5 antagonist is a compound
selected from the group consisting of: 605
10. The method of claim 1, wherein the DP-178 polypeptide consists
of an amino acid sequence in Table 3.
11. The method of claim 1, further comprising administering in
combination one or more antiviral or therapeutic agents useful for
the treatment of HIV.
12. The method of claim 11 wherein the antiviral agent is selected
from the group consisting of nucleoside reverse transcriptase
inhibitors, non-nucleoside reverse transcriptase inhibitors and
protease inhibitors.
13. The method of claim 11 wherein the antiviral agent is selected
from the group consisting of a DP-107 polypeptide, zidovudine,
lamivudine, zalcitabine, didanosine, stavudine, abacavir, adefovir
dipivoxil, lobucavir, BCH-10652, emitricitabine, beta-L-FD4, DAPD,
lodenosine, nevirapine, delaviridine, efavirenz, PNU-142721,
AG-1549, MKC442, (+)-calanolide A and B, saquinavir, indinavir,
ritonavir, nelfinavir, lasinavir, DMP-450, BMS-2322623, ABT-378,
amprenavir, hydroxyurea, ribavirin, IL-2, IL-12, Yissum No. 11607
and AG-1549.
14. A method of treating an HIV infection in an individual in need
of such treatment, comprising orally administering from 25 to 400
mg/day, one to two times per day, of a CCR5 antagonist of
structural formula 606or a pharmaceutically acceptable salt
thereof, and subcutaneously administering from 3 to 200 mg, or a
multiple thereof which reduces the viral load in the individual,
once two, three or four times per week, or once every other day, of
a T-20.
15. The method of claim 14, further comprising administering in
combination one or more antiviral or therapeutic agents useful in
the treatment of HIV.
16. The method of claim 15, wherein the antiviral agent is selected
from the group consisting of nucleoside reverse transcriptase
inhibitors, non-nucleoside reverse transcriptase inhibitors and
protease inhibitors.
17. The method of claim 15, wherein the antiviral agent is selected
from the group consisting of a DP-107 polypeptide, zidovudine,
lamivudine, zalcitabine, didanosine, stavudine, abacavir, adefovir
dipivoxil, lobucavir, BCH-10652, emitricitabine, beta-L-FD4, DAPD,
lodenosine, nevirapine, delaviridine, efavirenz, PNU-142721,
AG-1549, MKC442, (+)-calanolide A and B, saquinavir, indinavir,
ritonavir, nelfinavir, lasinavir, DMP450, BMS-2322623, ABT-378,
amprenavir, hydroxyurea, ribavirin, IL-2, IL-12, Yissum No. 11607
and AG-1549.
18. A kit comprising single package pharmaceutical compositions for
use in combination to treat HIV infection, which comprises in a
first container a pharmaceutical composition comprising a CCR5
antagonist, or pharmaceutically acceptable salt thereof, in a
pharmaceutically acceptable carrier, in an oral dosage from 25 to
600 mg to be administered from 1 to 3 times per week or every other
day, and in a second container a pharmaceutical composition
comprising a DP-178 polypeptide, or a pharmaceutically acceptable
derivative thereof, in a pharmaceutically acceptable carrier, in a
subcutaneous dosage from 3 to 200 mg, or a multiple thereof which
reduces the viral load by 1 or 2 logs.
19. The kit of claim 18 which comprises in additional container(s)
one or more pharmaceutical compositions comprising a
therapeutically effective amount of an antiviral or therepeutic
agent useful in the treatment of HIV in a pharmaceutically
acceptable carrier.
Description
FIELD OF THE INVENTION
[0001] This invention relates to combination therapies and to
methods for treating viral infections, e.g., human immunodeficiency
virus (HIV).
BACKGROUND OF THE INVENTION
[0002] The global health crisis caused by HIV, the causative agent
of Acquired Immunodeficiency Syndrome (AIDS), is unquestioned, and
while recent advances in drug therapies have been successful in
slowing the progression of AIDS, there is still a need to find a
safer, more efficient, less expensive way to control the virus.
[0003] HIV infection begins by attachment of the virus to a target
cell membrane through interaction with the cellular receptor CD4
and a secondary chemokine co-receptor molecule. It proceeds by
replication and dissemination of infected cells through the blood
and other tissue. There are various chemokine receptors, but for
macrophage-tropic HIV, believed to be the key pathogenic strain
that replicates in vivo in the early stages of infection, the
principal chemokine receptor required for the entry of HIV into the
cell is CCR5. It has also been reported that the CCR5 gene plays a
role in resistance to HIV infection, since individuals containing a
homozygous deletion for the CCR5 gene are largely resistant to HIV
infection. Therefore, interfering with the interaction between the
viral receptor CCR5 and HIV may block viral entry into the cell
without any significant side effects.
[0004] Attention has also been given to HIV-1 envelope proteins,
e.g., gp160, gp120 and gp41, which are major antigens for anti-HIV
antibodies present in AIDS patients. U.S. Pat. Nos. 5,464,933 and
6,020,459 and International Patent Publication WO 96/40191 describe
a portion of the HIV gp41 protein (known as DP-178, T-20, and
pentafuside), which is required for the virus to gain entry into
CD4.sup.+ cells. This protein fragment may prevent HIV from gaining
entry into host CD4.sup.+ cells and further prevent cell to cell
transmission of the virus.
[0005] T-20 has been administered as a continuous infusion or twice
daily (bid) subcutaneously, typically in dosages of 100 mg/day.
This greatly inconveniences the lifestyle of a patient receiving
this peptide therapy, since the patient undergoing this anti-HIV
therapy must visit a clinician twice a day to receive injections of
the peptide.
[0006] Vandamme et al. [Antiviral Chemistry & Chemotherapy
9:187-203 (1998)] disclose current clinical treatments of HIV-1
infections in man including at least triple drug combinations or
so-called Highly Active Antiretroviral Therapy ("HAART"); HAART
involves various combinations of nucleoside reverse transcriptase
inhibitors, non-nucleoside reverse transcriptase inhibitors and HIV
protease inhibitors. In compliant drug-naive patients, HAART is
effective in reducing mortality and progression of HIV-1 to AIDS.
However, these multidrug therapies do not eliminate HIV-1 and
long-term treatment usually results in multidrug resistance.
Furthermore, HIV therapies are expensive, costing tens of thousands
of dollars per year. It would be an advantage to provide an
anti-HIV therapy which blocks fusion and entry of HIV into
CD4.sup.+ cells by different mechanisms, has a more convenient
administration schedule, and as good if not a greater efficacy
against HIV compared with known treatments, having lower costs.
Thus, development of new drug therapies to provide better HIV-1
treatment remains a priority.
SUMMARY OF THE INVENTION
[0007] This invention provides an improved treatment for viral
infections, and, in particular, HIV infection. It provides novel
drug combination therapies, comprising an effective amount of a
first antiviral agent, which is a piperidine derivative of
structural formula I or II, or pharmaceutically acceptable salts
thereof, and an effective amount of a second antivrial agent which
is a DP-178 polypeptide, or a pharmaceutically acceptable
derivative thereof. A combination of the present invention is
administered to an individual infected with a virus, preferably
HIV, using a dosage and administration schedule adequate to inhibit
viral activity, viral expression, viral transmission, or reduce
viral load. A combination therapy of the present invention
preferably provides a therapy having improved efficacy over other
known anti-HIV combination therapies or therapies which contain
only one of the antiviral agents in the present combination. More
preferably, a combination therapy of the present invention is a
synergistic combination.
[0008] It is an advantage of this invention that a present
combination therapy allows the clinician to decrease the frequency
of administration of a DP-178 polypeptide (i.e., less than bid),
and, if desired, to administer the combination with one or more
additional therapeutic compounds as described, infra.
[0009] Thus, in one embodiment, the invention provides a method of
treating an HIV infection comprising administering in combination a
therapeutically effective amount of a CCR5 antagonist, or a
pharmaceutically acceptable salt thereof, and a therapeutically
effective amount of a DP-178 polypeptide, or a pharmaceutically
acceptable derivative thereof.
[0010] In another embodiment, the present invention provides a
method of treating an HIV infection comprising administering a CCR5
antagonist of structural formula 1
[0011] or a pharmaceutically acceptable salt thereof,
[0012] and T-20,
[0013] wherein the CCR5 antagonist is administered one to two times
a day in dosages of from 25 to 400 mg/day, and the T-20 is
administered once (qd) every other day or once (qd) two, three or
four times per week in a dosage of from 3 to 200 mg, or a multiple
thereof to reduce the viral load in the infected individual by 1 or
2 logs.
[0014] In another aspect, the present invention provides a kit
containing single package pharmaceutical compositions for use in
combination to treat HIV infection, which comprises in a first
container a pharmaceutical composition comprising a CCR5
antagonist, or pharmaceutically acceptable salt thereof, in a
pharmaceutically acceptable carrier, in an oral dosage from 25 to
600 mg to be administered from 1 to 3 times per week or every other
day, and in a second container a pharmaceutical composition
comprising a DP-178 polypeptide, or a pharmaceutically acceptable
derivative thereof, in a pharmaceutically acceptable carrier, in a
subcutaneous dosage from 3 to 200 mg, or a multiple thereof which
reduces the viral load by 1 or 2 logs.
DETAILED DESCRIPTION OF THE INVENTION
[0015] The term "viral infection" is used to describe a diseased
state, which can be latent, where a virus invades a cell, uses the
cell's reproductive machinery to multiply or replicate, and
ultimately lyses the cell causing cell death and release of progeny
virus particles followed by further infection of other cells by the
progeny.
[0016] The terms "treating" or "preventing" used in relation to a
viral infection means to inhibit viral activity, expression,
replication or transmission of a virus, or to prevent the virus
from establishing itself in a host cell, and which results in an
amelioration or alleviation of the symptoms of the disease caused
by the viral infection. A treatment or therapy is considered
therapeutic if there is a reduction in viral load or decrease in
mortality or morbidity.
[0017] A "therapeutically effective amount" of a DP-178 polypeptide
or a CCR5 antagonist compound, or their derivatives, is an amount
sufficient to treat or prevent a viral infection and according to a
suitable administration schedule, i.e., the amount and dosaging
schedule exhibits antiviral activity, thereby lowering HIV RNA
plasma levels in the serum of an infected individual to less than
500 copies per ml of serum, preferably to less than 200 copies per
ml of serum, more preferably to less than 50 copies per ml of
serum, and most prefereably the number of copies is undetectable,
as measured by quantitative, multi-cycle reverse transcriptase PCR
methodology. HIV RNA is preferably measured using the methodology
of Amplicor-1 Monitor 1.5 (available from Roche Diagnsotics) or of
Nuclisens HIV-1 QT-1.
[0018] The term "combination therapy" refers to a therapy for
treating viral infections, preferably HIV, which includes
administration of an effective amount of a CCR5 antagonist and a
DP-178 polypeptide. A combination therapy of this invention may
include one or more antiviral agents, e.g., HAART. In addition, a
combination therapy of this invention can be used as a prophylactic
measure in previously uninfected individuals after a possible acute
exposure to an HIV virus. Examples of such prophylactic use of the
peptides may include, but are not limited to, prevention of virus
transmission from mother to infant and other settings where the
likelihood of HIV transmission exists, such as, for example,
accidents in health care settings wherein workers are exposed to
HIV-containing blood products.
[0019] The term "synergistic" refers to a combination which is more
effective than the additive effects of any two or more single
agents. A "synergistic effect" refers to the ability to use lower
amounts or dosages of antiviral agents in a single therapy to treat
or prevent viral infection. The lower doses typically result in a
decreased toxicity without reduced efficacy. In addition, a
synergistic effect can improve efficacy, e.g., improved antiviral
activity, or avoid or reduce the extent of any viral resistance
against an antiviral agent. A synergistic effect between a DP-178
polypeptide, or a pharmaceutically acceptible derivative thereof,
and a CCR5 antagonist compound, or a pharmaceutically acceptable
salt thereof, can be determined from conventional antiviral assays,
e.g., as described infra. The results of an assay can be analyzed
using Chou and Talalay's combination method to obtain a Combination
Index (Chou and Talalay, 1984, Adv. Enzyme Regul. 22:27-55) and
`Dose Effect Analysis with Microcomputers` software (Chou and Chou,
1987, Software and Manual. p19-64. Elsevier Biosoft, Cambridge,
UK). A Combination Index value of less than 1 indicates synergy,
greater than 1 indicates antagonism and equal to 1 indicates an
additive effect. The results of these assays can also be analyzed
using the method of Pritchard and Shipman (Pritchard and Shipman,
1990, Antiviral Research 14: 181-206).
[0020] The term "pharmaceutically acceptable carrier" refers to a
carrier medium that does not interfere with the effectiveness of
the biological activity of the active ingredient, is chemically
inert and is generally not toxic to the recipient.
[0021] The term "pharmaceutically acceptable derivative" refers to
a tuncation, analog or other modification of a polypeptide, which
exhibits antiviral activity and is generally non-toxic.
[0022] The term "antiviral activity" refers to an inhibition of HIV
transmission to uninfected CD4.sup.+ cells, inhibition of the
replication of HIV, prevention of HIV from establishing itself in a
host, or ameliorating or alleviating the symptoms of the disease
caused by HIV infection. These effects can be evidenced by a
reduction in viral load or decrease in mortality and/or morbidity,
which assays are described infra. An antiviral agent, or anti-HIV-1
drug, has antiviral activity and is useful for treating HIV-1
infections alone, or as part of a multi-drug combination therapy,
e.g., the HAART triple and quadruple combination therapies.
[0023] A "therapeutic agent" is any molecule, compound or therapy
that improves the treatment of a viral infection or the diseases
caused thereby. Preferably, the therapeutic agent has antiviral
activity.
[0024] Viruses whose transmission may be inhibited by the antiviral
activity of a combination therapy of this invention include, for
example: human retroviruses, particularly HIV-1 and HIV-2 and the
human T-lymphocyte viruses (HTLV-I and II); non-human retroviruses,
including bovine leukosis virus, feline sarcoma and leukemia
viruses, simian immunodeficiency, sarcoma and leukemia viruses, and
sheep progress pneumonia viruses; non-retroviral viruses, including
human respiratory syncytial virus, canine distemper virus,
newcastle disease virus, human parainfluenza virus, influenza
viruses, measles viruses, Epstein-Barr viruses, hepatitis B
viruses, and simian Mason-Pfizer viruses; and non-enveloped
viruses, including picornaviruses such as polio viruses, hepatitis
A virus, enterovirus, echoviruses and coxsackie viruses,
papovaviruses such as papilloma virus, parvoviruses, adenoviruses
and reoviruses.
[0025] Piperidine Derivative CCR5 Antagonist Compounds
[0026] Compounds having the structural formulas I and II below, and
pharmaceutically acceptable salts thereof, are collectively
referred to herein as "CCR5 antagonists". These compounds
antagonize the CC chemokine receptor 5, and are described in U.S.
patent application Ser. Nos. 09/562,815 and 09/562,814 and in WO
00/66559 and WO 00/11632, which are each incorporated herein by
reference in their entireties.
[0027] In one embodiment, a combination therapy of this invention
comprises a compound of structural formula I, or a pharmaceutically
acceptable salt thereof: 2
[0028] wherein R.sup.6, X and R.sup.2 are as defined in Table
1:
1TABLE 2 R.sup.6 X R.sup.2 Br 3 4 Br 5 6 Br 7 8 Br 9 10 Br 11 12 Br
13 14 Br 15 16 Br 17 18 Br 19 20 Br 21 22 Br 23 24 Br 25 26
CH.sub.3SO.sub.2-- 27 28 Br 29 30 Br 31 32 Br 33 34 Br 35 36
H.sub.3CSO.sub.2-- 37 38 H.sub.3CSO.sub.2-- 39 40 F.sub.3C-- 41 42
H.sub.3CSO.sub.2-- 43 44 H.sub.3CSO.sub.2-- 45 46 F.sub.3CO-- 47 48
F.sub.3CO-- 49 50 F.sub.3CO-- 51 52 Br 53 54 Br 55 56 F.sub.3CO--
57 58 Br 59 60 Br 61 62 H.sub.3CSO.sub.2-- 63 64 H.sub.3CSO.sub.2--
65 66 H.sub.3CSO.sub.2-- 67 68 H.sub.3CSO.sub.2-- 69 70
H.sub.3CSO.sub.2-- 71 72 H.sub.3CSO.sub.2-- 73 74 F.sub.3C-- 75 76
F.sub.3CO-- 77 78 F.sub.3CO-- 79 80 Cl 81 82 Cl 83 84 Cl 85 86 Cl
87 88 Br 89 90 H.sub.3CSO.sub.2-- 91 92 F.sub.3C-- 93 94
H.sub.3CSO.sub.2-- 95 96 H.sub.3CSO.sub.2-- 97 98 F.sub.3C-- 99 100
F 101 102 F 103 104 F 105 106 Cl 107 108 F 109 110 Br 111 112 Br
113 114 Br 115 116 Br 117 118 F.sub.3C-- 119 120 Br 121 122 Br 123
124 Br 125 126 F.sub.3C-- 127 128 F.sub.3C-- 129 130 F.sub.3C-- 131
132 F 133 134 Br 135 136 Br 137 138 Br 139 140 Br 141 142 Br 143
144 F.sub.3CO-- 145 146 F.sub.3CO-- 147 148 F.sub.3CO-- 149 150
F.sub.3CO-- 151 152 F.sub.3CO-- 153 154 F.sub.3CO-- 155 156 Cl 157
158 Cl 159 160 Cl 161 162 Cl 163 164 F.sub.3CO-- 165 166 Br 167 168
Br 169 170 Br 171 172 Br 173 174 Br 175 176 Br 177 178 Br 179 180
Br 181 182 Bt 183 184 Br 185 186 Br 187 188 Br 189 190 Br 191 192
Br 193 194 Br 195 196 Br 197 198 Br 199 200 Br 201 202 Br 203 204
Br 205 206 Br 207 208 Br 209 210 Br 211 212 Br 213 214 Br 215 216
Br 217 218 Br 219 220 Br 221 222 Br 223 224 Br 225 226 Br 227 228
Br 229 230 Br 231 232 Br 233 234 F.sub.3C-- 235 236 Br 237 238 Br
239 240 Br 241 242 Br 243 244 Br 245 246 Br 247 248 Br 249 250 Br
251 252 Br 253 254 F.sub.3CO-- 255 256 F.sub.3CO-- 257 258 Br 259
260 Br 261 262 Br 263 264 Br 265 266 Br 267 268 Br 269 270
F.sub.3CO-- 271 272 F.sub.3CO-- 273 274 F.sub.3CO-- 275 276
F.sub.3CO-- 277 278 F.sub.3CO-- 279 280 F.sub.3CO-- 281 282 Cl 283
284 Cl 285 286 F.sub.3C-- 287 288 Cl 289 290 Cl 291 292 Cl 293 294
F.sub.3C-- 295 296 Cl 297 298 F.sub.3C-- 299 300 F.sub.3C-- 301 302
Cl 303 304 Cl 305 306 F.sub.3CO-- 307 308 F.sub.3CO-- 309 310
F.sub.3CO-- 311 312 F.sub.3CO-- 313 314 F.sub.3C-- 315 316
F.sub.3CO-- 317 318 F.sub.3C-- 319 320 F.sub.3C-- 321 322
F.sub.3C-- 323 324 F.sub.3CO-- 325 326 F.sub.3C-- 327 328
F.sub.3C-- 329 330 F.sub.3CO-- 331 332 F.sub.3CO-- 333 334
F.sub.3CO-- 335 336 F.sub.3C-- 337 338 F.sub.3CO-- 339 340
F.sub.3CO-- 341 342 F.sub.3C-- 343 344 F.sub.3C-- 345 346
F.sub.3CO-- 347 348 F.sub.3CO-- 349 350 F.sub.3CO-- 351 352
F.sub.3CO-- 353 354 F.sub.3CO-- 355 356 F.sub.3CO-- 357 358
F.sub.3CO-- 359 360 F.sub.3CO-- 361 362 F.sub.3CO-- 363 364
F.sub.3CO-- 365 366 F.sub.3CO-- 367 368 F.sub.3CO-- 369 370
F.sub.3CO-- 371 372 F.sub.3CO-- 373 374 Br 375 376 F.sub.3C-- 377
378 Br --CH.sub.2-- 379 Br --CH.sub.2-- 380 Br --CH.sub.2-- 381 Br
--CH.sub.2-- 382 Br --CH.sub.2-- 383 Br --CH.sub.2-- 384 Br 385 386
CH.sub.3SO.sub.2-- 387 388 Br 389 390 Br 391 392 F 393 394 F 395
396 F 397 398 Br 399 400 Cl 401 402 F.sub.3C-- 403 404
CH.sub.3SO.sub.2-- 405 406 CH.sub.3SO.sub.2-- 407 408 F.sub.3CO--
409 410 F.sub.3CO-- 411 412 CH.sub.3SO.sub.2-- 413 414
CH.sub.3SO.sub.2-- 415 416 F.sub.3C-- 417 418 F.sub.3CO-- 419 420
F.sub.3CO-- 421 422 F.sub.3C-- 423 424 H 425 426 F.sub.3CO-- 427
428 F.sub.3CO-- 429 430 F.sub.3CO-- 431 432 F.sub.3CO-- 433 434
F.sub.3CO-- 435 436 CH.sub.3SO.sub.2-- 437 438 CH.sub.3SO.sub.2--
439 440 CH.sub.3SO.sub.2-- 441 442 CH.sub.3SO.sub.2-- 443 444
CH.sub.3SO.sub.2-- 445 446 CH.sub.3SO.sub.2-- 447 448
CH.sub.3SO.sub.2-- 449 450 CH.sub.3SO.sub.2-- 451 452
[0029] In a specific embodiment, a combination therapy of this
invention comprises a compound of the structural formula 453
[0030] or a pharmaceutically acceptable salt thereof.
[0031] In another embodiment, a CCR5 antagonist compound used in a
combination therapy of this invention has the structural formula
II, or a pharmaceutically acceptable salt thereof: 454
[0032] wherein R, R.sup.3, R.sup.6 and R.sup.2 are as defined in
Table 2:
2TABLE 2 R R.sup.3 R.sup.6 R.sup.2 455 456 H 457 458 459 --CH.sub.3
460 461 462 H 463 464 465 H 466 467 468 H 469 470 471 H 472 473 474
--CH.sub.3 475 476 477 --CH.sub.3 478 479 480 --CH.sub.3 481 482
483 --CH.sub.3 484 485 486 --CH.sub.3 487 488 489 --CH.sub.3 490
491 492 H 493 494 495 H 496 497 498 --CH.sub.3 499 500 501
--CH.sub.3 502 503 504 --CH.sub.3 505 506 507 --CH.sub.3 508 509
510 --CH.sub.3 511 512 H --CH.sub.3 513 514 H --CH.sub.3 515 516 H
--CH.sub.3 517 518 H --CH.sub.3 519 520 521 --CH.sub.3 522 523 H H
524 525 526 H 527 528 529 --CH.sub.3 530 531 532 H 533 534 535
--CH.sub.3 536 537 538 H 539 540 541 H 542 543 544 H 545 546 547 H
548 549 550 --CH.sub.3 551 552 H --CH.sub.3 553 554 H --CH.sub.3
555 556 557 --CH.sub.2CH.sub.3 558 559 560 --CH.sub.2CH.sub.3 561
562 563 --CH.sub.2CH.sub.3 564 565 566 --CH.sub.3 567 568 569
--CH.sub.3 570 571 572 --CH.sub.3 573 574 575 --CH.sub.3 576 577
578 --CH.sub.3 579 580 581 --CH.sub.3 582 583 584 --CH.sub.3 585
586 587 --CH.sub.3 588 589 590 --CH.sub.3 591 592 593 --CH.sub.3
594 595 596 --CH.sub.3 597 598 599 --CH.sub.3 600 601 602
--CH.sub.3 603
[0033] In a specific embodiment, a combination therapy of this
invention comprises a compound of the structural formula: 604
[0034] or pharmaceutically acceptable salt thereof.
[0035] Certain CCR5 antagonist compounds used in this invention may
exist in different isomeric forms, e.g., enantiomers,
diastereoisomers and atropisomers. The invention contemplates all
such isomers both in pure form and in admixture, including racemic
mixtures.
[0036] Certain compounds will be acidic in nature, e.g., compounds
which possess a carboxyl or phenolic hydroxyl group. These
compounds may form pharmaceutically acceptable salts. Examples of
such salts include sodium, potassium, calcium, aluminum, gold and
silver salts. Also contemplated are salts formed with
pharmaceutically acceptable amines such as ammonia, alkyl amines,
hydroxyalkylamines, N-methylglucamine, and the like.
[0037] Certain basic compounds also form pharmaceutically
acceptable salts, e.g., acid addition salts. For example, the
pyrido-nitrogen atoms may form salts with strong acid, while
compounds having basic substituents such as amino groups also form
salts with weaker acids. Examples of suitable acids for salt
formation are hydrochloric, sulfuric, phosphoric, acetic, citric,
oxalic, malonic, salicylic, malic, fumaric, succinic, ascorbic,
maleic, methanesulfonic and other mineral and carboxylic acids well
known to those in the art. The salts are prepared by contacting the
free base form with a sufficient amount of the desired acid to
produce a salt in the conventional manner. The free base forms may
be regenerated by treating the salt with a suitable dilute aqueous
base solution such as dilute aqueous NaOH, potassium carbonate,
ammonia and sodium bicarbonate. The free base forms differ from
their respective salt forms somewhat in certain physical
properties, such as solubility in polar solvents, but the acid and
base salts are otherwise equivalent to their respective free base
forms for purposes of the invention.
[0038] All such acid and base salts are intended to be
pharmaceutically acceptable salts within the scope of the invention
and all acid and base salts are considered equivalent to the free
forms of the corresponding compounds for purposes of the
invention.
[0039] A CCR5 antagonist compound used in this invention can be
made according to procedures known in the art, e.g., using the
methods described in U.S. Pat. No. 5,883,096, U.S. Pat. No.
6,037,352, U.S. Pat. No. 5,889,006, U.S. Pat. No. 5,952,349, and
U.S. Pat. No. 5,977,138, which are incorporated herein by reference
in their entireties. U.S. patent application Ser. No. 09/562,815
and 09/562,814 and WO 00/66559 and WO 00/11632 provide specific
methodologies for making CCR5 antagonists used in this
invention.
[0040] DP-178 Polypeptides
[0041] DP-178 polypeptides (i.e., full-length DP-178 and
pharmaceutically acceptable derivatives thereof) are described in
U.S. Pat. No. 5,464,933, U.S. Pat. No. 6,020,459 and WO 96/40191,
which are each incorporated herein by reference in their
entireties. The DP-178 region of viral gp41 may mediate fusion of
HIV and the target cell membrane, by forming a complex with a
distal region on gp41 (possibly DP-107) needed for the virus to
fuse with the target cell. A DP-178 polypeptide used in this
invention exhibits antifusogenic activity, (inhibiting virus fusion
with a host cell membrane) antiviral activity, and/or the ability
to modulate intracellular processes involving coil-coil peptide
structures, and is identified or recognized by the ALLMOTI5,
107X178X4 and PLZIP search motifs described in U.S. Pat. No.
6,020,459.
[0042] The full-length DP-178 polypeptide is based upon 36 amino
acid residues spanning residues 638 to 673 of the transmembrane
protein gp41 from the HIV-1.sub.LAI isolate, and has an amino acid
sequence:
3 YTSLIHSLIEESQNQQEKNEQELLELDKWASLWNWF. (T-20; SEQ ID NO:1)
[0043] An analog of a DP-178 polypeptide used in this invention
contains one or more amino acid substitutions (conserved or
non-conserved), insertions and/or deletions of the full-length
DP-178 polypeptide. Homologs of DP-178 from other virus isolates or
species, or from other organisms, are also analogs of DP-178. In
addition to the full-length DP-178 polypeptide (SEQ ID NO:1), a
DP-178 polypeptide used in this invention includes truncations of
the full-length DP-178 comprising 3 to 35 contiguous amino acid
residues. DP-178 analogs can also be truncated. Truncations and
analogs of DP-178 are described in U.S. Pat. No. 5,464,933, U.S.
Pat. No. 6,020,459, and WO 96/40191.
[0044] An amino acid substitution in a DP-178 polypeptide used in
this invention can be a conserved or non-conserved substitution. A
conserved amino acid substitution replaces one or more amino acids
of a DP-178 polypeptide sequence with amino acids of similar
charge, size, and/or hydrophobicity characteristics, such as, for
example, a glutamic acid (E) to aspartic acid (D). A non-conserved
substitution replaces one or more amino acids of a DP-178
polypeptide sequence with amino acids possessing dissimilar charge,
size, and/or hydrophobicity characteristics, such as, for example,
a glutamic acid (E) to valine (V).
[0045] HIV-1 and HIV-2 enveloped proteins are structurally
distinct, but there exists a striking amino acid conservation
within the DP178-corresponding regions of HIV-1 and HIV-2. The
amino acid conservation is of a periodic nature, suggesting some
conservation of structure and/or function. Therefore, one class of
amino acid substitutions includes amino acid changes which are
predicted to stabilize the structure of DP-178 peptides of the
invention. Utilizing the DP-178 full-length and DP-178 analog
sequences described herein, and in U.S. Pat. No. 6,020,459, the
skilled artisan can readily compile DP-178 consensus sequences, and
ascertain the conserved amino acid residues which would represent
preferred amino acid substitutions.
[0046] An amino acid insertion in a DP-178 polypeptide used in this
invention includes one or more of a single amino acid residue or a
stretch of residues inserted into DP-178 polypeptide. The one or
more insertions can be made at the amino or carboxy terminus of a
DP-178 polypeptide or at an internal position of the polypeptide.
Preferably, an internal insertion is from 2 to 15 amino acids in
length. Preferably, an amino or carboxy terminus insertion is about
2 to about 50 amino acids and corresponds to a polypeptide sequence
of gp41 that is amino or carboxy to the full-length DP-178 amino
acid sequence.
[0047] A DP-178 polypeptide used in this invention can contain one
or more deletions. A deletion in a DP-178 polypeptide can be the
removal of one or more single amino acid residues or one or more of
a stretch of contiguous amino acids, and combinations thereof. A
DP-178 polypeptide containing a deletion is at least 4 to 6 amino
acids.
[0048] A DP-178 polypeptide homolog used in this invention is a
full-length or truncated DP-178 from a source other than
HIV-1.sub.LAI, e.g., other HIV-1 and HIV-2 isolates,
non-HIV-1.sub.LAI enveloped viruses, non-enveloped viruses and
non-viral organisms. A DP-178 homolog can also contain one or more
deletions, insertions or substitutions, as described above. A
DP-178 homolog can comprise, e.g., peptide sequences present in
transmembrane proteins of enveloped viruses or polypeptide
sequences present in non-enveloped and non-viral organisms.
Homologs of DP-178 are described in U.S. Pat. No. 5,464,933, U.S.
Pat. No. 6,020,459, and WO 96/40191, and include:
4 YTNTIYTLLEESQNQQEKNEQELLELDKWASLWNWF; (DP-185; SEQ ID NO:3)
YTGIIYNLLEESQNQQEKNEQELLELDKWANLWNWF; (SEQ ID NO:4)
YTSLIYSLLEKSQIQQEKNEQELLELDKWASLWNWF; (SEQ ID NO:5) and
LEANISQSLEQAQIQQEKNMYELQKLNSWDVFT- NWL, (SEQ ID NO:6)
[0049] which are derived from HIV-1.sub.SF2, HIV-1.sub.RF,
HIV-1.sub.MN, and HIV-2.sub.NIHZ isolates, respectively.
[0050] A DP-178 polypeptide used in this invention can have a
modification to the amino and/or carboxy terminus to provide, e.g.,
increased bioavailability, stability, or reduced host immune
recognition. For example, the terminal NH.sub.2 group can be
replaced by a hydrophobic group, carbobenzyl, dansyl, or
t-butoxycarbonyl, an acetyl group, or a 9-fluorenylmethoxy-carbonyl
(FMOC) group. A macromolecular group, e.g., a lipid-fatty acid
conjugate, polyethylene glycol, carbohydrate or peptide group, can
be covalently attached to the NH.sub.2 or to the modification. The
terminal COOH group can be replaced, e.g., by an amido group, a
t-butoxycarbonyl group, or a covalently attached macromolecular
group, e.g., a lipid-fatty acid conjugate, polyethylene glycol,
carbohydrate or an additional polypeptide group. A macromolecular
group can be attached to the COOH or the modification.
[0051] The following Table provides examples of DP-178 polypeptides
that can be used in the present invention:
5TABLE 3 YTSLIHSLIEESQNQQEKNEQELLELDKWASLWNWF; (SEQ ID NO:1)
YTNTIYTLLEESQNQQEKNEQELLELDKWASLWNWF; (DP-185; SEQ ID NO:3)
YTGIIYNLLEESQNQQEKNEQELLELDKWANLWNWF; (SEQ ID NO:4)
YTSLIYSLLEKSQIQQEKNEQELLELDKWASLWNWF; (SEQ ID NO:5)
LEANISQSLEQAQIQQEKNMYELQKLNSWDVFTNWL; (SEQ ID NO:6)
CGGNNLLRAIEAQQHLLQLTVWGIKQLQARILAVERYLKDQ; (SEQ ID NO:7)
WMEWDREINNYTSLIGSLIEESQNQQEKNEQELLE; (SEQ ID NO:8)
YTSLIHSLIEESQNQQEKNEQELLELDKWASLWNWFNITNWLWLIKFI; (SEQ ID NO:9)
YTSLIHSLIEESQNQQEKNEQELLELDKWASLWNAF; (SEQ ID NO:10)
YTSLIHSLIEESQNQQEKNEQELLELDKWASLANWF; (SEQ ID NO:11)
YTSLIHSLIEESQNQQEKNEQQLLELDKWASLWNWF; (SEQ ID NO:12)
YTSLIHSLIEESQNQQEKNEQELLQLDKWASLWNWF; (SEQ ID NO:13)
YTSLIHSLIEESQNQQEKNQQELLQLDKWASLWNWF; (SEQ ID NO:14)
YTSLIHSLQEESQNQQEKNEQELLELDKWASLWNWF; (SEQ ID NO:15)
YTSLIHSLIEESQNQQEKNEQELLELDKWASLWNW; (SEQ ID NO:16)
YTSLIHSLIEQSQNQQEKNEQELLELDKWASLWNWF; (SEQ ID NO:17)
YTSLIHSLIQESQNQQEKNEQELLELDKWASLWNWF; (SEQ ID NO:18)
YTSLIHSLIEESQNQQEKNEQQLLELDKWASLWNWF; (SEQ ID NO:19)
YTSLIQSLIEESQNQQEKNEQQLLELDKWASLWNWF; (SEQ ID NO:20)
YTSLIHSLIEESQNQQEKNEQELLELDKWASLFNFF; (SEQ ID NO:21)
YTSLIHSLIEESQNLQEKNEQELLELDKWASLWNWF; (SEQ ID NO:22)
YTSLIHSLIEESQNQQEKLEQELLELDKWASLWNWF; (SEQ ID NO:23)
YTSLIHSLIEESQNQQEKNEQELLEFDKWASLWNWF; (SEQ ID NO:24)
YTSLIHSLIEESQNQQEKNEQELLELDKWASPWNWF; (SEQ ID NO:25)
YTSLIHSLIEESQNQQEKNEQELLELDKWASLWNSF; (SEQ ID NO:26)
NKSLEQIWNNMTWMEWDREINNYTSLIHSLIEESQNQQEKNEQELLELDKASLWNWF; (SEQ ID
NO:27) SLEQIWNNMTWMEWDREINNYTSLIHSLIEESQNQQ; (SEQ ID NO:28)
LEQIWNNMTWMEWDREINNYTSLIHSLIEESQNQQE; (SEQ ID NO:29)
EQIWNNMTWMEWDREINNYTSLIHSLIEESQNQQEK; (SEQ ID NO:30)
QIWNNMTWMEWDREINNYTSLIHSLIEESQNQQEKN; (SEQ ID NO:31)
IWNNMTWMEWDREINNYTSLIHSLIEESQNQQEKNE; (SEQ ID NO:32)
WNNMTWMEWDREINNYTSLIHSLIEESQNQQEKNEQ; (SEQ ID NO:33)
NNMTWMEWDREINNYTSLIHSLIEESQNQQEKNEQE; (SEQ ID NO:34)
NMTWMEWDREINNYTSLIHSLIEESQNQQEKNEQEL; (SEQ ID NO:35)
MTWMEWDREINNYTSLIHSLIEESQNQQEKNEQELLELDKWASLWNW; (SEQ ID NO:36)
TWMEWDREINNYTSLIHSLIEESQNQQEKNEQELLE; (SEQ ID NO:37)
WMEWDREINNYTSLIHSLIEESQNQQEKNEQELLEL; (SEQ ID NO:38)
WMEWDREINNYTSLIHSLIEESQNQQEKNEQELLE; (SEQ ID NO:39)
MEWDREINNYTSLIHSLIEESQNQQEKNEQELLELD; (SEQ ID NO:40)
EWDREINNYTSLIHSLIEESQNQQEKNEQELLELDK; (SEQ ID NO:41)
WDREINNYTSLIHSLIEESQNQQEKNEQELLELDKW; (SEQ ID NO:42)
DREINNYTSLIHSLIEESQNQQEKNEQELLELDKWA; (SEQ ID NO:43)
REINNYTSLIHSLIEESQNQQEKNEQELLELDKWAS; (SEQ ID NO:44)
EINNYTSLIHSLIEESQNQQEKNEQELLELDKWASL; (SEQ ID NO:45)
INNYTSLIHSLIEESQNQQEKNEQELLELDKWASLW; (SEQ ID NO:46)
NYTSLIHSLIEESQNQQEKNEQELLELDKWASLWNW; (SEQ ID NO:47)
TSLIHSLIEESQNQQEKNEQELLELDKWASLWNWFN; (SEQ ID NO:48)
SLIHSLIEESQNQQEKNEQELLELDKWASLWNWFNI; (SEQ ID NO:49)
LIHSLIEESQNQQEKNEQELLELDKWASLWNWFNIT; (SEQ ID NO:50)
TSLIHSLIEESQNQQEKNEQELLELDKWASLWNWF; (SEQ ID NO:51)
LIHSLIEESQNQQEKNEQELLELDKWASLWNWF; (SEQ ID NO:52)
ESQNQQEKNEQELLELDKWASLWNWF; (SEQ ID NO:53) NQQEKNEQELLELDKWASLWNWF;
(SEQ ID NO:54) EKNEQELLELDKWASLWNWF; (SEQ ID NO:55)
LRAIEAQQHLLQLTVWQIKQLQARILAV; (SEQ ID NO:56)
YTSLIYSLLEKSQIQQEKNEQELLELDKWASLWNWF; (SEQ ID NO:57)
NNLLRAIEAQQGLLQLTVWGIKQLQARILAVERYLKDQ; (SEQ ID NO:58)
NNLLRAIEAQQHLLQLTVWGIKQLQARILAVERYLKDQGGC; (SEQ ID NO:59)
NNLLRAIEAQQHLLQLTVWGIKQLQARILAVERYLKDQGGC; (SEQ ID NO:60)
CGGNNLLRAIEAQQHLLQLTVWGIKQLQARILAVERYKDQGGC; (SEQ ID NO:61) and
LSGIVQQQNNLLRAIEAQQHLLQLTVWGIKQLQARILAV. (SEQ ID NO:62)
DP-107 Peptides
[0052] Optionally, a DP-178 polypeptide can be used with a DP-107
polypeptide, which is described in U.S. Pat. No. 6,020,459 and in
WO 96/40191. Since DP-107 and DP-178 portions of the transmembrane
protein gp41 form non-covalent protein-protein interactions which
is necessary for normal viral infectivity, the combination of a
DP-178 and DP-107 peptides may form a complex with viral gp41 and
interfere with the fusogenic process. Thus, it may be desirable to
use a DP-107 polypeptide in a combination therapy of this
invention.
[0053] A full-length DP-107 polypeptide is 38 amino acids
corresponding to residues 558 to 595 of HIV-1.sub.LAI transmembrane
gp41 protein, as shown:
6 NNLLRAIEAQQHLLQLTVWQIKQLQARILAVERYLKDQ. (SEQ ID NO:2)
[0054] A DP-107 polypeptide used in this invention includes
truncations of the full-length DP-107 polypeptide, and contains
from 3 to 38 contiguous amino acid residues. Examples of DP-107
truncations are described in U.S. Pat. No. 6,020,459 and WO
96/40191.
[0055] A DP-107 polypeptide used in this invention includes analogs
of the full-length and of truncated DP-107, which analogs contain
one or more amino acid substitutions (conserved or non-conserved),
insertions and/or deletions, as described above for DP-178. Analogs
of DP-107 are described in U.S. Pat. No. 6,020,459 and WO 96/40191.
A DP-107 polypeptide can also contain a modification at the amino
or carboxy terminus to provide, e.g., increased bioavailability,
stability, or reduced host immune recognition, as described above
for DP-178 and in U.S. Pat. No. 6,020,459 and WO 96/40191. Homologs
of DP-107 from other virus isolates or species, or from other
organisms, are also analogs of DP-107, and are described in U.S.
Pat. No. 6,020,459 and WO 96/40191.
[0056] A DP-107 polypeptide used in this invention, whether
full-length, truncated, an analog (i.e., containing an insertion,
deletion, substitution, or a homolog of DP-107), or otherwise
modified, exhibits antiviral activity, antifusogenic activity, or
modulates intracellular processes involving coil-coil peptides, and
has structural and/or amino acid motif similarity to DP-107, and
can be identified or recognized by the ALLMOTI5, 107X178X4 and
PLZIP search motifs described in detail in U.S. Pat. No.
6,020,459.
[0057] Preparation of Polypeptides
[0058] A polypeptide used in this invention can be synthesized or
prepared by techniques well known in the art. See, e.g., Creighton,
1983, Proteins: Structures and Molecular Principles, W.H. Freeman
and Co., NY. Short peptides, for example, can be synthesized on a
solid support or in solution. Longer peptides can be made using
recombinant DNA techniques. The nucleotide sequences encoding the
peptides of the invention may be synthesized, and/or cloned, and
expressed according to techniques well known in the art. See, e.g.,
Sambrook, et al., 1989, Molecular Cloning, A Laboratory Manual,
Vols. 1-3, Cold Spring Harbor Press, NY.
[0059] A polypeptide used in this invention can be synthesized in a
manner such that one or more of the bonds which link the amino acid
residues of the peptides are non-peptide bonds, as described supra.
A non-peptide bond can be formed by conventional reactions well
known in the art, e.g., by imino, ester, hydrazide, semicarbazide,
and azo bonds. Peptides used in the invention can also be
synthesized having additional chemical groups (i.e., modifications,
as described supra) present at the amino and/or carboxy termini,
e.g., to enhance the stability, bioavailability, and/or inhibitory
activity of the polypeptide according to conventional
methodologies.
[0060] A polypeptide used in this invention can also be synthesized
with an altered steric configuration, e.g., the D-isomer of one or
more of the amino acid residues of the polypeptide is used versus
the L-isomer. If desired, one or more amino acid residues of a
polypeptide used in this invention can be substituted by a
non-naturally occurring amino acid residue. Alterations such as
these may serve to increase the stability, bioavailability and/or
inhibitory action of the peptides of the invention.
[0061] Specific methods for synthesizing a DP-178 or DP-107
polypeptide used in this invention are described in U.S. Pat. No.
6,015,881, which use solid and liquid phase synthesis procedures to
synthesize and combine groups of specific polypeptide fragments to
yield the peptide of interest. Generally, these methods involve
synthesizing specific side-chain protected peptide fragment
intermediates of a peptide on a solid support, coupling the
protected fragments in solution to form a protected peptide,
followed by deprotection of the side chains to yield the final
polypeptide.
[0062] Use of Additional Therapeutic Agents
[0063] A combination therapy of the present invention can further
include one or more, preferably one to three, additional antiviral
agents useful in anti-HIV therapy. Antivirals which act on a
different target molecule involved in viral replication, which act
on a different target molecule involved in viral transmission,
which prevent or reduce the occurrence of viral resistance, and
which act on a different loci of the same molecule, can be used
with a combination therapy of the invention. Such classes of agents
include those which cause an inhibition of viral reverse
transcriptase, inhibition of viral mRNA capping, inhibition of the
HIV protease, inhibition of protein glycosylation, inhibition of
viral fusion (e.g., DP-107 as described above), and other antiviral
drugs, some of which are discussed herein, not falling within the
aforementioned acitivities. One skilled in the art can determine a
wide variety of antiviral therapies using an antiviral agent that
exhibits one of these modes of activity. In particular, the
combinations known as HAART can be used with a combination of this
invention. Preferably, lower doses of antiviral agents are used in
a combination therapy of the invention, thus reducing the toxicity
associated with that agent, without loss of antiviral activity and
while reducing or avoiding viral resistance.
[0064] The term "nucleoside and nucleotide reverse transcriptase
inhibitors" ("NRTI"s) as used herein means nucleosides and
nucleotides and analogues thereof that inhibit the activity of
HIV-1 reverse transcriptase, which catalyzes the conversion of
viral genomic HIV-1 RNA into proviral HIV-1 DNA. Nucleoside
derivatives include but are not limited to 2',3'-dideoxyadenosine
(ddA); 2',3'-dideoxyguanosine (ddG); 2',3'-dideoxyinosine (ddI);
2',3'-dideoxycytidine (ddC); 2',3'-dideoxythymidine (ddT);
2',3'-dideoxy-dideoxythymidine (d4T) and
3'-azido-2',3'-dideoxythymidine (AZT).
[0065] Alternatively, halogenated nucleoside derivatives can be
used, preferably 2',3'-dideoxy-2'-fluoronucleosides, e.g.,
2',3'-dideoxy-2'-fluoroadenosine; 2',3'-dideoxy-2'-fluoroinosine;
2',3'-dideoxy-2'-fluorothymidine; 2',3'-dideoxy-2'-fluorocytosine;
and 2',3'-dideoxy-2',3'-didehydro-2'-fluoronucleosides including,
but not limited to 2',3'-dideoxy-2',3'-didehydro-2'-fluorothymidine
(Fd4T). Preferably, the 2',3'-dideoxy-2'-fluoronucleosides of the
invention are those in which the fluorine linkage is in the beta
configuration, e.g., 2',3'-dideoxy-2'-beta-fluoroadenosine (F-ddA),
2',3'-dideoxy-2'-beta-fluo- roinosine (F-ddI), and
2',3'-dideoxy-2'-beta-fluorocytosine (F-ddC).
[0066] Typical suitable NRTIs include zidovudine (AZT) available
under the RETROVIR tradename from Glaxo-Wellcome Inc., (Research
Triangle, N.C.); didanosine (ddI) available under the VIDEX
tradename from Bristol-Myers Squibb Co. (Princeton, N.J.);
zalcitabine (ddC) available under the HIVID tradename from Roche
Pharmaceuticals, (Nutley, N.J.); stavudine (d4T) available under
the ZERIT trademark from Bristol-Myers Squibb Co.; lamivudine (3TC)
available under the EPIVIR tradename from Glaxo-Wellcome; abacavir
(1592U89) disclosed in WO96/30025 and available under the ZIAGEN
trademark; adefovir dipivoxil [bis(POM)-PMEA] available under the
PREVON tradename from Gilead Sciences (Foster City, Calif.);
lobucavir (BMS-180194), a nucleoside reverse transcriptase
inhibitor disclosed in EP-0358154 and EP-0736533 and under
development by Bristol-Myers Squibb, (Princeton, N.J.); BCH-10652,
a reverse transcriptase inhibitor (in the form of a racemic mixture
of BCH-10618 and BCH-10619) under development by Biochem Pharma,
(Laval, Quebec, Canada); emitricitabine [(-)-FTC] licensed from
Emory University's U.S. Pat. No. 5,814,639 and under development by
Triangle Pharmaceuticals, (Durham, N.C.); beta-L-FD4 (also called
beta-L-D4C and named beta-L-2',3'-dicleoxy-5-fluoro-cytidene)
licensed by Yale University to Vion Pharmaceuticals, (New Haven
Conn.); DAPD, the purine nucleoside, (-)-beta-D-2,6,-diamino-purine
dioxolane disclosed in EP 0656778 and licensed by Emory University
and the University of Georgia to Triangle Pharmaceuticals; and
lodenosine (FddA), 9-(2,3-dideoxy-2-fluoro-b-D-threo-
-pentofuranosyl)adenine, an acid stable purine-based reverse
transcriptase inhibitor discovered by the NIH and under development
by U.S. Bioscience Inc., (West Conshohoken, Pa.).
[0067] The term "non-nucleoside reverse transcriptase inhibitors"
("NNRTI"s) as used herein means non-nucleosides that inhibit the
activity of HIV-1 reverse transcriptase. Typical suitable NNRTIs
include nevirapine (BI-RG-587) available under the VIRAMUNE
tradename from Boehringer Ingelheim, the manufacturer for Roxane
Laboratories (Columbus Ohio); delaviradine (BHAP, U-90152)
available under the RESCRIPTOR tradename from Pharmacia &
Upjohn Co. (Bridgewater N.J.); efavirenz (DMP-266) a
benzoxazin-2-one disclosed in WO94/03440 and available under the
SUSTIVA tradename from DuPont Pharmaceutical Co. (Wilmington Del.);
PNU-142721, a furopyridine-thio-pyrimide under development by
Pharmacia & Upjohn Co.; AG-1549 (formerly Shionogi # S-1153);
5-(3,5-dichlorophenyl)--
thio-4-isopropyl-1-(4-pyridyl)methyl-IH-imidazol-2-ylmethyl
carbonate disclosed in WO 96/10019 and under clinical development
by Agouron Pharmaceuticals, Inc. (LaJolla Calif.); MKC-442
(1-(ethoxy-methyl)-5-(1-m-
ethylethyl)-6-(phenylmethyl)-(2,4(1H,3H)-pyrimidinedione)
discovered by Mitsubishi Chemical Co. and under development by
Triangle Pharmaceuticals; and (+)-calanolide A (NSC-675451) and B,
coumarin derivatives disclosed in U.S. Pat. No. 5,489,697 licensed
to Med Chem Research, which is co-developing (+) calanolide A with
Vita-Invest as an orally administrated product.
[0068] The term "protease inhibitor" describes an antiviral agent
that inhibits the HIV-1 protease, which is required for the
proteolytic cleavage of viral polyprotein precursors (e.g., viral
GAG and GAG Pol polyproteins), into individual functional proteins
found in infectious HIV-1. PIs may work primarily during or after
virus assembly (i.e., viral budding) to inhibit the maturation of
virions into a mature infectious state. HIV protease inhibitors
include compounds having a peptidomimetic structure, high molecular
weight (7600 daltons) and substantial peptide character, e.g.,
CRIXIVAN (available from Merck) as well as nonpeptide protease
inhibitors, e.g., VIRACEPT (available from Agouron).
[0069] Typical suitable PIs include saquinavir (Ro 31-8959)
available in hard gel capsules under the INVIRASE tradename and as
soft gel capsules under the FORTOVASE tradename from Roche
Pharmaceuticals; ritonavir (ABT-538) available under the NORVIR
tradename from Abbott Laboratories (Abbott Park, Ill.); indinavir
(MK-639) available under the CRIXIVAN tradename from Merck &
Co., Inc., (West Point, Pa.); nelfnavir (AG-1343) available under
the VIRACEPT tradename from Agouron Pharmaceuticals, Inc.;
amprenavir (141W94), tradename AGENERASE, a non-peptide protease
inhibitor under development by Vertex Pharmaceuticals, Inc.,
(Cambridge, Mass.) and available from Glaxo-Wellcome under an
expanded access program; lasinavir (BMS-234475) available from
Bristol-Myers Squibb (discovered by Novartis (CGP-61755); DMP450, a
cyclic urea discovered by Dupont and under development by Triangle
Pharmaceuticals; BMS-2322623, an azapeptide under development by
Bristol-Myers Squibb, as a second generation HIV-1 PI; ABT-378
under development by Abbott; and AG-1549, an orally active
imidazole carbamate discovered by Shionogi (Shionogi #S-1153) and
under development by Agouron Pharmaceuticals, Inc.
[0070] A combination therapy of the present invention can be used
in further combination with uridine phosphorylase inhibitors, e.g.,
acyclouridine compounds, including benzylacyclouridine (BAU),
benzyloxybenzylacyclouridine (BBAU), aminomethylbenzylacyclouridine
(AMBAU), aminomethylbenzyloxybenzylacyclouridine (AMB-BAU),
hydroxymethylbenzylacyclouridine (HMBAU), and
hydroxymethylbenzyloxybenzy- lacyclouridine (HMBBAU); cytokines or
cytokine inhibitors, e.g., IL-2 [PROLEUKIN (aldesleukin), Chiron
Corp. (Emeryville, Calif.) described in EP-0142268, EP-0176299, RE
33653, U.S. Pat. No. 4,530,787, U.S. Pat. No. 4,569,790, U.S. Pat.
No. 4,604,377, U.S. Pat. No. 4,748,234, U.S. Pat. No. 4,752,585,
and U.S. Pat. No. 4,949,314], IL-12 (disclosed in WO96/25171, Roche
Pharmaceuticals and American Home Prodocts, Madison, N.J.),
IFN-.alpha. (INTRON-A; Schering Plough), IFN-.beta. and
IFN-.gamma., and recombinant forms thereof, TNF-.alpha. inhibitors,
and MNX-160; viral capping drugs which interfere with 5'-mRNA
processing, e.g., Ribavirin
(1-.beta.-D-ribofuranosyl-1H-1,2,4-triazole-3-carboxamide- ;
described in U.S. Pat. No. 4,211,771 ICN Pharmaceuticals, Inc.,
Costa Mesa, Calif.;); antiviral agents active against a variety of
lipid-enveloped viruses including HIV, e.g., Amphotericin B or its
methyl ester (Fungizone; Gibco) as a lipid binding molecule with
antiviral (anti-HIV) activity; inhibitors of glycoprotein
processing, e.g., castonospermine (Boehringer Mannheim);
hydroxyurea (Droxia; Bristol-Myers Squibb), which is an inhibitor
of ribonucleoside triphosphate reductase, shown in preclinical
studies to have a synergistic effect on the activity of didanosine,
and can be used with stavudine; Yissum Project No. 11607, which is
a synthetic protein based on the HIV-1 Vif protein, under
preclinical development by Yissum Research Development Co.
(Jerusalem, Israel).
[0071] Antifungal agents and other agents against opportunistic
infections, including TB, HBV, EBV, and CMV, can be used with
combination therapy of the present invention for alleviating or
treating a disease associated with HIV-infected, immunosuppressed
patients
[0072] Preferred additional therapeutic agents used in a
combination therapy of this invention include ddI, DP-107, 3TC,
ribavirin, and IFN-.beta..
[0073] Antiviral Activity Assays
[0074] In vitro assays for the study of antiviral compounds active
at different stages of HIV infection (acute, co-cultivation, and
chronic) are well known in the art, e.g., see Lambert et al.,
Antiviral Res. 21: 327-342, (1993). These assays can be used to
assess the effects of a combination therapy of this invention with
additional therapeutic agents if desired. The antiviral activity
exhibited by antiviral agents used in a combination therapy of this
invention can be measured using in vitro assays, e.g., as described
below, which test a DP-178 polypeptide's ability to inhibit
syncytia formation or inhibit infection by cell-free virus, and/or
test the CCR5 inhibitory and antagonistic activity of a CCR5
antagonist compound used in this invention. Using these assays,
such parameters as the relative antiviral activity of the peptides,
exhibit against a given strain of virus and/or the strain specific
inhibitory activity of the peptide can be determined.
[0075] Furthermore, the effectiveness of the present combination
therapy can be determined by the multiple drug analysis method of
Chou and Talalay (Chou and Talalay, 1984, Adv. Enzyme Regul.
22:27-55) and `Dose-Effect Analysis with Microcomputer Software
(Chou and Chou, 1987, Software and Manual. p. 19-64. Elsevier
Biosoft, Cambridge, UK). Analysis of drug-drug interactions,
including synergy and antagonism between antiviral agnets used in a
combination therapy of this invention, can be calculated by the
MacSynergy computer program (Pritchard and Shipman, 1990, Antiviral
Research 14: 181-206).
[0076] CCR5 antagonist compounds of the invention inhibit RANTES
("regulated upon activation, normal T cell expressed secreted",
which is a natural ligand of CCR5) binding with activity (Ki) from
about 0.1 to 2000 nM, preferably from 0.1 to 1000 nM, more
preferably from about 0.1 to 500 nM, and most preferably from about
0.1 to 100 nM.
CCR5 Membrane Binding Assay
[0077] A high throughput screen utilizing a CCR5 membrane binding
assay identifies inhibitors of RANTES binding. This assay utilizes
membranes prepared from NIH 3T3 cells expressing the human CCR5
chemokine receptor which have the ability to bind to RANTES. Using
a 96-well plate format, membrane preparations are incubated with
.sup.125I-RANTES in the presence or absence of compound for one
hour. Compounds are serially diluted over a wide range of 0.001
.mu.g/ml to 1 .mu.g/ml and tested in triplicates. Reaction
cocktails are harvested through glass fiber filters, and washed
thoroughly. Total counts for replicates are averaged and data
reported as the concentration required to inhibit 50 percent of
total .sup.125I-RANTES binding. Compounds with potent activity in
the membrane binding assay are further characterized in secondary
cell-based HIV-1 entry and replication assays.
HIV-1 Entry Assay
[0078] Replication defective HIV-1 reporter virions are generated
by cotransfection of a plasmid encoding the NL4-3 strain of HIV-1
(which has been modified by mutation of the envelope gene and
introduction of a luciferase reporter plasmid) along with a plasmid
encoding one of several HIV-1 envelope genes as described in Connor
et al, Virology 206: 935-944 (1995). Following transfection of the
two plasmids by calcium phosphate precipitation, the viral
supernatants can be harvested on day 3 and a functional viral titer
determined. These stocks are then used to infect U87 cells stably
expressing CD4 and the chemokine receptor CCR5, which have been
preincubated with or without test compound. Infections are carried
out for 2 hours at 37.degree. C., the cells washed and media
replaced with fresh media containing a CCR5 antagonist compound.
The cells are incubated for 3 days, lysed and luciferase activity
determined. Results are reported as the concentration of compound
required to inhibit 50% of the luciferase activity in the control
cultures.
HIV-1 Replication Assay
[0079] This assay uses primary peripheral blood mononuclear cells
or the stable U87-CCR5 cell line to determine the effect of
antiviral agents used in the present combination therapy to block
infection of primary HIV-1 strains. The primary lymphocytes are
purified from normal healthy donors and stimulated in vitro with
PHA and IL-2 three days prior to infection. Using a 96-well plate
format, cells are pretreated with drug for 1 hour at 37.degree. C.
and subsequently infected with an M-tropic HIV-1 isolate. Following
infection, the cells are washed to remove residual inoculum and
cultured in the presence of compound for 4 days. Culture
supernatants are harvested and viral replication measured by
determination of viral p24 antigen concentration.
Calcium Flux Assay
[0080] Cells expressing the HIV coreceptor CCR5 are loaded with
calcium sensitive dyes prior to addition of compound or the natural
CCR5 ligand (RANTES). Compounds with agonist properties will induce
a calcium flux signal in the cell, while CCR5 antagonists are
identified as compounds which do not induce signaling by themselves
but are capable of blocking signaling by the natural ligand
RANTES.
GTP.gamma.S Binding Assay (Secondary Membrane Binding Assay)
[0081] A GTP.gamma.S binding assay measures receptor activation by
CCR5 ligands. This assay measures the binding of .sup.35S
labeled-GTP to receptor coupled G-proteins, which occurs as a
result of receptor activation by an appropriate ligand. In this
assay, the CCR5 ligand RANTES is incubated with membranes from CCR5
expressing cells and binding to the receptor (or activation) is
determined by assaying for bound .sup.35S label. The assay
quantitatively determines if compounds exhibit agonist
characteristics by inducing activation of the receptor or
alternatively antagonist properties by measuring inhibition of
RANTES binding in a competitive or non-competitive fashion.
Chemotaxis Assay
[0082] The chemotaxis assay is a functional assay which
characterizes the agonist vs. antagonist properties of the test
CCR5 antagonist compounds. The assay measures the ability of a
non-adherent murine cell line expressing human CCR5 (BaF-550) to
migrate across a membrane in response to either test compounds or
natural ligands (i.e., RANTES, MIP-1.beta.). Cells migrate across
the permeable membrane towards compounds with agonist activity.
Compounds that are antagonists not only fail to induce chemotaxis,
but are also capable of inhibiting cell migration in response to
known CCR5 ligands.
Cell Fusion Assay
[0083] Assays for detecting cell fusion events are well known in
the art, and can be used to determine the ability of a DP-178
polypeptide and/or CCR5 antagonist to inhibit membrane fusion or
viral-induced syncytial. Cell fusion assays are generally performed
in vitro, by culturing cells which, in the absence of any
treatment, would typically undergo an observable level of syncytial
formation. For example, uninfected cells (e.g., CD4.sup.+ cells
such as Molt or MEM cells) are incubated in the presence of cells
chronically infected with a virus (e.g. HIV) that induces cell
fusion or in the presence or absence of a polypeptide to be
assayed. After incubation (e.g. 24 hours at 37.degree. C.) the cell
culture is examined microscopically for the presence of
multinucleated giant cells, which are indicative of cell fusion and
syncytial formation. Well known stains, such as crystal violet
stain, can be used to facilitate the visualization of syncytial
formation.
[0084] Pharmaceutical Compositions, Dosaging and Administration
Schedules
[0085] Since the DP-178 site of action is at the surface of the
virus, and prevents free virus from infecting host cells and cell
to cell transmission of the virus, the use of DP-178 in combination
with a CCR5 antagonist compound, which blocks the host cell
receptor for HIV, provides different therapeutic targets having
mechanisms of action that can provide an additive or synergistic
effect. Preferably, a combination of the present invention is used
at lower concentrations of the antiviral agents, resulting in
decreased toxicity. Thus, a combination therapy of this invention
may not only reduce the effective dose of a drug required for
antiviral activity, thereby reducing its toxicity, but may also
improve the absolute antiviral effect as a result of attacking the
virus through multiple mechanisms. The combinations of the present
invention also provide a means for circumventing or decreasing the
chance of development of viral resistance.
[0086] Administration of a DP-178 polypeptide, or a
pharmaceutically acceptable derivative thereof, and a CCR5
antagonist compound, or a pharmaceutically acceptable salt thereof,
"in combination" includes procedures in which both agents are
administered together as an admixture (single dosage form), and
also procedures in which the two agents are administered separately
but simultaneously, e.g., through separate intravenous lines or
oral and intravenous administration.
[0087] Administration "in combination" further includes sequential
administrations of a DP-178 polypeptide and CCR5 antagonist
compound in separate administrations such that one of the drugs is
given first, followed by the second, e.g., as in cycling therapy
(i.e., the administration of a first antiviral compound for a
period of time, followed by the administration of a second
antiviral agent for a period of time and repeating this sequential
administration to reduce the development of resistance to one of
the compounds).
[0088] A pharmaceutical composition suitable for use in the present
invention is formulated using a therapeutically effective amount of
the antiviral agents to achieve their intended purpose with a
suitable pharmacological carrier. For all such purposes, the exact
formulation, route of administration and dosage can be chosen by
the clinician in view of the patient's condition (see, e.g., Fingl
et al., 1975, in "The Pharmacological Basis of Therapeutics", Ch.
1).
[0089] Procedures for formulation and administration may be found
in "Remington's Pharmaceutical Sciences", 18th ed., 1990, Mack
Publishing Co., Easton, Pa. Pharmaceutical compositions for use in
accordance with the present invention thus may be formulated in
conventional manner using one or more physiologically acceptable
carriers comprising excipients and auxiliaries which facilitate
processing of the active compounds into preparations which can be
used pharmaceutically. Proper formulation is dependent upon the
route of administration chosen. Routes of administration include
oral, rectal, transmucosal, intestinal, parenteral, intramuscular,
subcutaneous, intramedullary, intrathecal, direct intraventricular,
intravenous (i.e., injection or continuous infusion),
intraperitoneal, intranasal, inhalation, intraocular, transdermal,
topical, vaginal, and the like.
[0090] A pharmaceutical preparation used in a combination therapy
of the present invention is preferably in a unit dosage form. In
such form, the preparation is subdivided into suitably sized unit
doses containing an appropriate amount of the antiviral agent.
Effective dosages of the peptides of the invention to be
administered may be determined through procedures well known to
those in the art which address such parameters as biological
half-life, bioavailability, and toxicity.
[0091] Dosage forms include but are not limited to tablets,
dispersions, powders, suspensions, suppositories, solutions,
emulsions, capsules, dragee cores, aerosols, implants, creams,
patches, minipumps, and the like. Most preferably, administration
of a DP-178 polypeptide is intravenous. Most preferably,
administration of a CCR5 antagonist compound is oral.
[0092] An amount of a CCR5 antagonist, or derivative thereof, in a
unit dose preparation, particularly for oral administration, can
vary from about 10 mg to about 500 mg. The actual dosage of a CCR5
antagonist can be varied depending upon such factors as age,
condition and size of the patient, severity of the symptoms being
treated, and type of HIV isolate, which can be determined by one
skilled in the art. The total daily dosage can be divided and
administered in portions during a day of administration as needed.
A typical recommended total daily dosage regimen can range from
about 20 mg to about 800 mg, preferably 25 mg to 600 mg, more
preferably about 50 mg to 400 mg, most preferably 100 mg to 200 mg
per day of administration.
[0093] In the combination therapy of this invention, a
therapeutically effective amount of a CCR5 antagonist, or
derivative thereof, can be administered daily or less than daily,
e.g., two to four times per week or alternatively every other day.
The dosage can also be divided into two to four doses, or a single
dose can be provided. Preferably, a therapeutically effective
amount of a CCR5 antagonist, or derivative thereof, is administered
orally each day, twice per day (bid). The practitioner may
determine that the efficacy of the present combination allows less
frequent administration of a CCR5 antagonist, e.g., once per day or
less frequent, e.g., from 1 to 3 times per week or every other
day.
[0094] An amount of a DP-178 polypeptide, or a pharmaceutically
acceptable derivative thereof, is administered, in a unit dose
preparation can be administered parenterally, e.g., as a continuous
infusion or injection. Doses of a DP-178 polypeptide can range from
3 mg to 200 mg (or a multiple thereof to reduce viral load by 1 to
2 logs), preferably from 7 mg to 100 mg, more preferably from 20 mg
to 35 mg, per day of administration. It is an advantage of the
present invention that the polypeptide can be administered less
often than twice per day (bid), which was previously reported for
the use of T-20 in clinical trials. Thus, a combination of the
present invention permits administration of a therapeutically
effective amount of DP-178 polypeptide once per day (qid),
preferably once (qd) two to four times per week, more preferably
once (qd) every other day.
[0095] The doses and dosage regimens of the NRTIs, NNRTIs, Pls and
other therapeutic agents used in combination with a therapy of this
invention can be determined by the attending clinician in view of
the approved doses and dosage regimens in the package inserts or as
set forth in the protocols for the particular agent, taking into
consideration the age, sex and condition of the patient, the
severity of the condition treated and the HIV isolate.
[0096] Typical suitable multidrug combination therapies such as (i)
at least three anti-HIV-1 drugs selected from two NRTIs, one PI, a
second PI, and one NNRTI; and (ii) at least two antiviral agents
selected from NNRTIs and PIs. Typical suitable HAART-multidrug
combination therapies include:
[0097] (a) triple combination therapies such as two NRTIs and one
PI; or
[0098] (b) two NRTIs and one NNRTI; and
[0099] (c) quadruple combination therapies such as two NRTIs, one
PI and a second PI or one NNRTI.
[0100] These multidrug combinations can be used in combination with
a CCR5 antagonist and a DP-178 polypeptide, or their
pharmaceutically acceptable salts/derivatives.
[0101] In treatment of naive patients, it is preferred to start
anti-HIV-1 treatment with the triple multidrug combination therapy
with a CCR5 antagonist and a DP-178 polypeptide, or their
pharmaceutically acceptable salts/derivatives; the use of two NRTIs
and one PI is prefered unless there is intolerance to PIs. Drug
compliance is essential. The CD.sup.4+ and HIV-1-RNA plasma levels
should be monitored every 3-6 months. Should viral load plateau, an
additional drug, e.g., one PI or one NNRTI can be added. Typical
therapies for use in combination with the present invention are
further described in the following table:
7 ANTIVIRAL MULTI DRUG COMBINATION THERAPIES A. Triple Combination
Therapies 1. Two NRTIs.sup.1 + one PI.sup.2 2. Two NRTIs.sup.1 +
one NNRTI.sup.3 B. Quadruple Combination Therapies.sup.4 Two NRTIs
+ one PI + a second PI or one NNRTI C. Alternatives:.sup.5 Two
NRTI.sup.1 One NRTI.sup.5 + one PI.sup.2 Two PIs.sup.6 + one
NRTI.sup.7 or NNRTI.sup.3 One PI.sup.2 + one NRTI.sup.7 + one
NNRTI.sup.3 Footnotes to Table .sup.1One of the following:
zidovudine + lamivudine; zidovudine + didanosine; stavudine +
lamivudine; stavudine + didanosine; zidovudine + zalcitabine.
.sup.2Indinavir, nelfinavir, ritonavir or saquinavir soft gel
capsules. .sup.3Nevirapine or delavirdine. .sup.4See Vandamne et
al., Antiviral Chemistry & Chemotherapy 9:193-197 (1998).
.sup.5Alternative regimens are for patients unable to take a
recommended regimen because of compliance problems or toxicity, and
for those who fail or relapse on a recommended regimen. Double
nucleoside combinations may lead to HIV-resistance and clinical
failure in many patients. .sup.6Most data obtained with saquinavir
and ritonavir (each 400 mg bid). .sup.7Zidovudine, stavudine or
didanosine.
[0102]
Sequence CWU 1
1
62 1 36 PRT Human immunodeficiency virus type 1 1 Tyr Thr Ser Leu
Ile His Ser Leu Ile Glu Glu Ser Gln Asn Gln Gln 1 5 10 15 Glu Lys
Asn Glu Gln Glu Leu Leu Glu Leu Asp Lys Trp Ala Ser Leu 20 25 30
Trp Asn Trp Phe 35 2 38 PRT Human immunodeficiency virus type 1 2
Asn Asn Leu Leu Arg Ala Ile Glu Ala Gln Gln His Leu Leu Gln Leu 1 5
10 15 Thr Val Trp Gln Ile Lys Gln Leu Gln Ala Arg Ile Leu Ala Val
Glu 20 25 30 Arg Tyr Leu Lys Asp Gln 35 3 36 PRT Human
immunodeficiency virus type 1 3 Tyr Thr Asn Thr Ile Tyr Thr Leu Leu
Glu Glu Ser Gln Asn Gln Gln 1 5 10 15 Glu Lys Asn Glu Gln Glu Leu
Leu Glu Leu Asp Lys Trp Ala Ser Leu 20 25 30 Trp Asn Trp Phe 35 4
36 PRT Human immunodeficiency virus type 1 4 Tyr Thr Gly Ile Ile
Tyr Asn Leu Leu Glu Glu Ser Gln Asn Gln Gln 1 5 10 15 Glu Lys Asn
Glu Gln Glu Leu Leu Glu Leu Asp Lys Trp Ala Asn Leu 20 25 30 Trp
Asn Trp Phe 35 5 36 PRT Human immunodeficiency virus type 1 5 Tyr
Thr Ser Leu Ile Tyr Ser Leu Leu Glu Lys Ser Gln Ile Gln Gln 1 5 10
15 Glu Lys Asn Glu Gln Glu Leu Leu Glu Leu Asp Lys Trp Ala Ser Leu
20 25 30 Trp Asn Trp Phe 35 6 36 PRT Human immunodeficiency virus
type 1 6 Leu Glu Ala Asn Ile Ser Gln Ser Leu Glu Gln Ala Gln Ile
Gln Gln 1 5 10 15 Glu Lys Asn Met Tyr Glu Leu Gln Lys Leu Asn Ser
Trp Asp Val Phe 20 25 30 Thr Asn Trp Leu 35 7 41 PRT Human
immunodeficiency virus type 1 7 Cys Gly Gly Asn Asn Leu Leu Arg Ala
Ile Glu Ala Gln Gln His Leu 1 5 10 15 Leu Gln Leu Thr Val Trp Gly
Ile Lys Gln Leu Gln Ala Arg Ile Leu 20 25 30 Ala Val Glu Arg Tyr
Leu Lys Asp Gln 35 40 8 35 PRT Human immunodeficiency virus type 1
8 Trp Met Glu Trp Asp Arg Glu Ile Asn Asn Tyr Thr Ser Leu Ile Gly 1
5 10 15 Ser Leu Ile Glu Glu Ser Gln Asn Gln Gln Glu Lys Asn Glu Gln
Glu 20 25 30 Leu Leu Glu 35 9 48 PRT Human immunodeficiency virus
type 1 9 Tyr Thr Ser Leu Ile His Ser Leu Ile Glu Glu Ser Gln Asn
Gln Gln 1 5 10 15 Glu Lys Asn Glu Gln Glu Leu Leu Glu Leu Asp Lys
Trp Ala Ser Leu 20 25 30 Trp Asn Trp Phe Asn Ile Thr Asn Trp Leu
Trp Leu Ile Lys Phe Ile 35 40 45 10 36 PRT Human immunodeficiency
virus type 1 10 Tyr Thr Ser Leu Ile His Ser Leu Ile Glu Glu Ser Gln
Asn Gln Gln 1 5 10 15 Glu Lys Asn Glu Gln Glu Leu Leu Glu Leu Asp
Lys Trp Ala Ser Leu 20 25 30 Trp Asn Ala Phe 35 11 36 PRT Human
immunodeficiency virus type 1 11 Tyr Thr Ser Leu Ile His Ser Leu
Ile Glu Glu Ser Gln Asn Gln Gln 1 5 10 15 Glu Lys Asn Glu Gln Glu
Leu Leu Glu Leu Asp Lys Trp Ala Ser Leu 20 25 30 Ala Asn Trp Phe 35
12 36 PRT Human immunodeficiency virus type 1 12 Tyr Thr Ser Leu
Ile His Ser Leu Ile Glu Glu Ser Gln Asn Gln Gln 1 5 10 15 Glu Lys
Asn Glu Gln Gln Leu Leu Glu Leu Asp Lys Trp Ala Ser Leu 20 25 30
Trp Asn Trp Phe 35 13 36 PRT Human immunodeficiency virus type 1 13
Tyr Thr Ser Leu Ile His Ser Leu Ile Glu Glu Ser Gln Asn Gln Gln 1 5
10 15 Glu Lys Asn Glu Gln Glu Leu Leu Gln Leu Asp Lys Trp Ala Ser
Leu 20 25 30 Trp Asn Trp Phe 35 14 36 PRT Human immunodeficiency
virus type 1 14 Tyr Thr Ser Leu Ile His Ser Leu Ile Glu Glu Ser Gln
Asn Gln Gln 1 5 10 15 Glu Lys Asn Gln Gln Glu Leu Leu Gln Leu Asp
Lys Trp Ala Ser Leu 20 25 30 Trp Asn Trp Phe 35 15 36 PRT Human
immunodeficiency virus type 1 15 Tyr Thr Ser Leu Ile His Ser Leu
Gln Glu Glu Ser Gln Asn Gln Gln 1 5 10 15 Glu Lys Asn Glu Gln Glu
Leu Leu Glu Leu Asp Lys Trp Ala Ser Leu 20 25 30 Trp Asn Trp Phe 35
16 35 PRT Human immunodeficiency virus type 1 16 Tyr Thr Ser Leu
Ile His Ser Leu Ile Glu Glu Ser Gln Asn Gln Gln 1 5 10 15 Glu Lys
Asn Glu Gln Glu Leu Leu Glu Leu Asp Lys Trp Ala Ser Leu 20 25 30
Trp Asn Trp 35 17 36 PRT Human immunodeficiency virus type 1 17 Tyr
Thr Ser Leu Ile His Ser Leu Ile Glu Gln Ser Gln Asn Gln Gln 1 5 10
15 Glu Lys Asn Glu Gln Glu Leu Leu Glu Leu Asp Lys Trp Ala Ser Leu
20 25 30 Trp Asn Trp Phe 35 18 36 PRT Human immunodeficiency virus
type 1 18 Tyr Thr Ser Leu Ile His Ser Leu Ile Gln Glu Ser Gln Asn
Gln Gln 1 5 10 15 Glu Lys Asn Glu Gln Glu Leu Leu Glu Leu Asp Lys
Trp Ala Ser Leu 20 25 30 Trp Asn Trp Phe 35 19 36 PRT Human
immunodeficiency virus type 1 19 Tyr Thr Ser Leu Ile His Ser Leu
Ile Glu Glu Ser Gln Asn Gln Gln 1 5 10 15 Glu Lys Asn Glu Gln Gln
Leu Leu Glu Leu Asp Lys Trp Ala Ser Leu 20 25 30 Trp Asn Trp Phe 35
20 36 PRT Human immunodeficiency virus type 1 20 Tyr Thr Ser Leu
Ile Gln Ser Leu Ile Glu Glu Ser Gln Asn Gln Gln 1 5 10 15 Glu Lys
Asn Glu Gln Gln Leu Leu Glu Leu Asp Lys Trp Ala Ser Leu 20 25 30
Trp Asn Trp Phe 35 21 36 PRT Human immunodeficiency virus type 1 21
Tyr Thr Ser Leu Ile His Ser Leu Ile Glu Glu Ser Gln Asn Gln Gln 1 5
10 15 Glu Lys Asn Glu Gln Glu Leu Leu Glu Leu Asp Lys Trp Ala Ser
Leu 20 25 30 Phe Asn Phe Phe 35 22 36 PRT Human immunodeficiency
virus type 1 22 Tyr Thr Ser Leu Ile His Ser Leu Ile Glu Glu Ser Gln
Asn Leu Gln 1 5 10 15 Glu Lys Asn Glu Gln Glu Leu Leu Glu Leu Asp
Lys Trp Ala Ser Leu 20 25 30 Trp Asn Trp Phe 35 23 36 PRT Human
immunodeficiency virus type 1 23 Tyr Thr Ser Leu Ile His Ser Leu
Ile Glu Glu Ser Gln Asn Gln Gln 1 5 10 15 Glu Lys Leu Glu Gln Glu
Leu Leu Glu Leu Asp Lys Trp Ala Ser Leu 20 25 30 Trp Asn Trp Phe 35
24 36 PRT Human immunodeficiency virus type 1 24 Tyr Thr Ser Leu
Ile His Ser Leu Ile Glu Glu Ser Gln Asn Gln Gln 1 5 10 15 Glu Lys
Asn Glu Gln Glu Leu Leu Glu Phe Asp Lys Trp Ala Ser Leu 20 25 30
Trp Asn Trp Phe 35 25 36 PRT Human immunodeficiency virus type 1 25
Tyr Thr Ser Leu Ile His Ser Leu Ile Glu Glu Ser Gln Asn Gln Gln 1 5
10 15 Glu Lys Asn Glu Gln Glu Leu Leu Glu Leu Asp Lys Trp Ala Ser
Pro 20 25 30 Trp Asn Trp Phe 35 26 36 PRT Human immunodeficiency
virus type 1 26 Tyr Thr Ser Leu Ile His Ser Leu Ile Glu Glu Ser Gln
Asn Gln Gln 1 5 10 15 Glu Lys Asn Glu Gln Glu Leu Leu Glu Leu Asp
Lys Trp Ala Ser Leu 20 25 30 Trp Asn Ser Phe 35 27 57 PRT Human
immunodeficiency virus type 1 27 Asn Lys Ser Leu Glu Gln Ile Trp
Asn Asn Met Thr Trp Met Glu Trp 1 5 10 15 Asp Arg Glu Ile Asn Asn
Tyr Thr Ser Leu Ile His Ser Leu Ile Glu 20 25 30 Glu Ser Gln Asn
Gln Gln Glu Lys Asn Glu Gln Glu Leu Leu Glu Leu 35 40 45 Asp Lys
Ala Ser Leu Trp Asn Trp Phe 50 55 28 36 PRT Human immunodeficiency
virus type 1 28 Ser Leu Glu Gln Ile Trp Asn Asn Met Thr Trp Met Glu
Trp Asp Arg 1 5 10 15 Glu Ile Asn Asn Tyr Thr Ser Leu Ile His Ser
Leu Ile Glu Glu Ser 20 25 30 Gln Asn Gln Gln 35 29 36 PRT Human
immunodeficiency virus type 1 29 Leu Glu Gln Ile Trp Asn Asn Met
Thr Trp Met Glu Trp Asp Arg Glu 1 5 10 15 Ile Asn Asn Tyr Thr Ser
Leu Ile His Ser Leu Ile Glu Glu Ser Gln 20 25 30 Asn Gln Gln Glu 35
30 36 PRT Human immunodeficiency virus type 1 30 Glu Gln Ile Trp
Asn Asn Met Thr Trp Met Glu Trp Asp Arg Glu Ile 1 5 10 15 Asn Asn
Tyr Thr Ser Leu Ile His Ser Leu Ile Glu Glu Ser Gln Asn 20 25 30
Gln Gln Glu Lys 35 31 36 PRT Human immunodeficiency virus type 1 31
Gln Ile Trp Asn Asn Met Thr Trp Met Glu Trp Asp Arg Glu Ile Asn 1 5
10 15 Asn Tyr Thr Ser Leu Ile His Ser Leu Ile Glu Glu Ser Gln Asn
Gln 20 25 30 Gln Glu Lys Asn 35 32 36 PRT Human immunodeficiency
virus type 1 32 Ile Trp Asn Asn Met Thr Trp Met Glu Trp Asp Arg Glu
Ile Asn Asn 1 5 10 15 Tyr Thr Ser Leu Ile His Ser Leu Ile Glu Glu
Ser Gln Asn Gln Gln 20 25 30 Glu Lys Asn Glu 35 33 36 PRT Human
immunodeficiency virus type 1 33 Trp Asn Asn Met Thr Trp Met Glu
Trp Asp Arg Glu Ile Asn Asn Tyr 1 5 10 15 Thr Ser Leu Ile His Ser
Leu Ile Glu Glu Ser Gln Asn Gln Gln Glu 20 25 30 Lys Asn Glu Gln 35
34 36 PRT Human immunodeficiency virus type 1 34 Asn Asn Met Thr
Trp Met Glu Trp Asp Arg Glu Ile Asn Asn Tyr Thr 1 5 10 15 Ser Leu
Ile His Ser Leu Ile Glu Glu Ser Gln Asn Gln Gln Glu Lys 20 25 30
Asn Glu Gln Glu 35 35 36 PRT Human immunodeficiency virus type 1 35
Asn Met Thr Trp Met Glu Trp Asp Arg Glu Ile Asn Asn Tyr Thr Ser 1 5
10 15 Leu Ile His Ser Leu Ile Glu Glu Ser Gln Asn Gln Gln Glu Lys
Asn 20 25 30 Glu Gln Glu Leu 35 36 47 PRT Human immunodeficiency
virus type 1 36 Met Thr Trp Met Glu Trp Asp Arg Glu Ile Asn Asn Tyr
Thr Ser Leu 1 5 10 15 Ile His Ser Leu Ile Glu Glu Ser Gln Asn Gln
Gln Glu Lys Asn Glu 20 25 30 Gln Glu Leu Leu Glu Leu Asp Lys Trp
Ala Ser Leu Trp Asn Trp 35 40 45 37 36 PRT Human immunodeficiency
virus type 1 37 Thr Trp Met Glu Trp Asp Arg Glu Ile Asn Asn Tyr Thr
Ser Leu Ile 1 5 10 15 His Ser Leu Ile Glu Glu Ser Gln Asn Gln Gln
Glu Lys Asn Glu Gln 20 25 30 Glu Leu Leu Glu 35 38 36 PRT Human
immunodeficiency virus type 1 38 Trp Met Glu Trp Asp Arg Glu Ile
Asn Asn Tyr Thr Ser Leu Ile His 1 5 10 15 Ser Leu Ile Glu Glu Ser
Gln Asn Gln Gln Glu Lys Asn Glu Gln Glu 20 25 30 Leu Leu Glu Leu 35
39 35 PRT Human immunodeficiency virus type 1 39 Trp Met Glu Trp
Asp Arg Glu Ile Asn Asn Tyr Thr Ser Leu Ile His 1 5 10 15 Ser Leu
Ile Glu Glu Ser Gln Asn Gln Gln Glu Lys Asn Glu Gln Glu 20 25 30
Leu Leu Glu 35 40 36 PRT Human immunodeficiency virus type 1 40 Met
Glu Trp Asp Arg Glu Ile Asn Asn Tyr Thr Ser Leu Ile His Ser 1 5 10
15 Leu Ile Glu Glu Ser Gln Asn Gln Gln Glu Lys Asn Glu Gln Glu Leu
20 25 30 Leu Glu Leu Asp 35 41 36 PRT Human immunodeficiency virus
type 1 41 Glu Trp Asp Arg Glu Ile Asn Asn Tyr Thr Ser Leu Ile His
Ser Leu 1 5 10 15 Ile Glu Glu Ser Gln Asn Gln Gln Glu Lys Asn Glu
Gln Glu Leu Leu 20 25 30 Glu Leu Asp Lys 35 42 36 PRT Human
immunodeficiency virus type 1 42 Trp Asp Arg Glu Ile Asn Asn Tyr
Thr Ser Leu Ile His Ser Leu Ile 1 5 10 15 Glu Glu Ser Gln Asn Gln
Gln Glu Lys Asn Glu Gln Glu Leu Leu Glu 20 25 30 Leu Asp Lys Trp 35
43 36 PRT Human immunodeficiency virus type 1 43 Asp Arg Glu Ile
Asn Asn Tyr Thr Ser Leu Ile His Ser Leu Ile Glu 1 5 10 15 Glu Ser
Gln Asn Gln Gln Glu Lys Asn Glu Gln Glu Leu Leu Glu Leu 20 25 30
Asp Lys Trp Ala 35 44 36 PRT Human immunodeficiency virus type 1 44
Arg Glu Ile Asn Asn Tyr Thr Ser Leu Ile His Ser Leu Ile Glu Glu 1 5
10 15 Ser Gln Asn Gln Gln Glu Lys Asn Glu Gln Glu Leu Leu Glu Leu
Asp 20 25 30 Lys Trp Ala Ser 35 45 36 PRT Human immunodeficiency
virus type 1 45 Glu Ile Asn Asn Tyr Thr Ser Leu Ile His Ser Leu Ile
Glu Glu Ser 1 5 10 15 Gln Asn Gln Gln Glu Lys Asn Glu Gln Glu Leu
Leu Glu Leu Asp Lys 20 25 30 Trp Ala Ser Leu 35 46 36 PRT Human
immunodeficiency virus type 1 46 Ile Asn Asn Tyr Thr Ser Leu Ile
His Ser Leu Ile Glu Glu Ser Gln 1 5 10 15 Asn Gln Gln Glu Lys Asn
Glu Gln Glu Leu Leu Glu Leu Asp Lys Trp 20 25 30 Ala Ser Leu Trp 35
47 36 PRT Human immunodeficiency virus type 1 47 Asn Tyr Thr Ser
Leu Ile His Ser Leu Ile Glu Glu Ser Gln Asn Gln 1 5 10 15 Gln Glu
Lys Asn Glu Gln Glu Leu Leu Glu Leu Asp Lys Trp Ala Ser 20 25 30
Leu Trp Asn Trp 35 48 36 PRT Human immunodeficiency virus type 1 48
Thr Ser Leu Ile His Ser Leu Ile Glu Glu Ser Gln Asn Gln Gln Glu 1 5
10 15 Lys Asn Glu Gln Glu Leu Leu Glu Leu Asp Lys Trp Ala Ser Leu
Trp 20 25 30 Asn Trp Phe Asn 35 49 36 PRT Human immunodeficiency
virus type 1 49 Ser Leu Ile His Ser Leu Ile Glu Glu Ser Gln Asn Gln
Gln Glu Lys 1 5 10 15 Asn Glu Gln Glu Leu Leu Glu Leu Asp Lys Trp
Ala Ser Leu Trp Asn 20 25 30 Trp Phe Asn Ile 35 50 36 PRT Human
immunodeficiency virus type 1 50 Leu Ile His Ser Leu Ile Glu Glu
Ser Gln Asn Gln Gln Glu Lys Asn 1 5 10 15 Glu Gln Glu Leu Leu Glu
Leu Asp Lys Trp Ala Ser Leu Trp Asn Trp 20 25 30 Phe Asn Ile Thr 35
51 35 PRT Human immunodeficiency virus type 1 51 Thr Ser Leu Ile
His Ser Leu Ile Glu Glu Ser Gln Asn Gln Gln Glu 1 5 10 15 Lys Asn
Glu Gln Glu Leu Leu Glu Leu Asp Lys Trp Ala Ser Leu Trp 20 25 30
Asn Trp Phe 35 52 33 PRT Human immunodeficiency virus type 1 52 Leu
Ile His Ser Leu Ile Glu Glu Ser Gln Asn Gln Gln Glu Lys Asn 1 5 10
15 Glu Gln Glu Leu Leu Glu Leu Asp Lys Trp Ala Ser Leu Trp Asn Trp
20 25 30 Phe 53 26 PRT Human immunodeficiency virus type 1 53 Glu
Ser Gln Asn Gln Gln Glu Lys Asn Glu Gln Glu Leu Leu Glu Leu 1 5 10
15 Asp Lys Trp Ala Ser Leu Trp Asn Trp Phe 20 25 54 23 PRT Human
immunodeficiency virus type 1 54 Asn Gln Gln Glu Lys Asn Glu Gln
Glu Leu Leu Glu Leu Asp Lys Trp 1 5 10 15 Ala Ser Leu Trp Asn Trp
Phe 20 55 20 PRT Human immunodeficiency virus type 1 55 Glu Lys Asn
Glu Gln Glu Leu Leu Glu Leu Asp Lys Trp Ala Ser Leu 1 5 10 15 Trp
Asn Trp Phe 20 56 28 PRT Human immunodeficiency virus type 1 56 Leu
Arg Ala Ile Glu Ala Gln Gln His Leu Leu Gln Leu Thr Val Trp 1 5 10
15 Gln Ile Lys Gln Leu Gln Ala Arg Ile Leu Ala Val 20 25 57 36 PRT
Human immunodeficiency virus type 1 57 Tyr Thr Ser Leu Ile Tyr Ser
Leu Leu Glu Lys Ser Gln Ile Gln Gln 1 5 10 15 Glu Lys Asn Glu Gln
Glu Leu Leu Glu Leu Asp Lys Trp Ala Ser Leu 20 25 30 Trp Asn Trp
Phe 35 58 38 PRT Human immunodeficiency virus type 1 58 Asn Asn Leu
Leu Arg Ala Ile Glu Ala Gln
Gln Gly Leu Leu Gln Leu 1 5 10 15 Thr Val Trp Gly Ile Lys Gln Leu
Gln Ala Arg Ile Leu Ala Val Glu 20 25 30 Arg Tyr Leu Lys Asp Gln 35
59 41 PRT Human immunodeficiency virus type 1 59 Asn Asn Leu Leu
Arg Ala Ile Glu Ala Gln Gln His Leu Leu Gln Leu 1 5 10 15 Thr Val
Trp Gly Ile Lys Gln Leu Gln Ala Arg Ile Leu Ala Val Glu 20 25 30
Arg Tyr Leu Lys Asp Gln Gly Gly Cys 35 40 60 41 PRT Human
immunodeficiency virus type 1 60 Asn Asn Leu Leu Arg Ala Ile Glu
Ala Gln Gln His Leu Leu Gln Leu 1 5 10 15 Thr Val Trp Gly Ile Lys
Gln Leu Gln Ala Arg Ile Leu Ala Val Glu 20 25 30 Arg Tyr Leu Lys
Asp Gln Gly Gly Cys 35 40 61 43 PRT Human immunodeficiency virus
type 1 61 Cys Gly Gly Asn Asn Leu Leu Arg Ala Ile Glu Ala Gln Gln
His Leu 1 5 10 15 Leu Gln Leu Thr Val Trp Gly Ile Lys Gln Leu Gln
Ala Arg Ile Leu 20 25 30 Ala Val Glu Arg Tyr Lys Asp Gln Gly Gly
Cys 35 40 62 39 PRT Human immunodeficiency virus type 1 62 Leu Ser
Gly Ile Val Gln Gln Gln Asn Asn Leu Leu Arg Ala Ile Glu 1 5 10 15
Ala Gln Gln His Leu Leu Gln Leu Thr Val Trp Gly Ile Lys Gln Leu 20
25 30 Gln Ala Arg Ile Leu Ala Val 35
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